Vaccines containing attenuated bacteria

Abstract

The invention relates to a vaccine comprising a bacterium attenuated by a non-reverting mutation in a gene encoding a protein which promotes folding of extracytoplasmic proteins. Such mutations were initially identified as being useful in vaccines from a bank of randomly inserted, transposon mutants in which attenuation was determined as a reduction in virulence of the organism in the mouse model of infection. Site directed mutation of the gene results in a strain which shows at least 4 logs of attenuation when delivered both orally and intravenously. Animals vaccinated with such a strain are protected against subsequent challenge with the parent wild type strain. Finally, heterologous antigens such as the non-toxic and protective, binding domain from tetanus toxin, fragment C, can be delivered via the mucosal immune system using such strains of bacteria. This results in the induction of a fully protective immune response to subsequent challenge with native tetanus toxin.

Description

[0001]This is a continuation of International Application No. PCT / GB98 / 03680, filed Dec. 10, 1998, the contents of which are hereby incorporated by referee.BACKGROUND OF THE INVENTION[0002]The principle behind vaccination is to induce an immune response in the host thus providing protection against subsequent challenge with a pathogen. This may be achieved by inoculation with a live attenuated strain of the pathogen (i.e. a strain having reduced virulence such that it does not cause the disease caused by the virulent pathogen).[0003]Classically, live attenuated vaccine strains of bacteria and viruses have been selected using one of two different methodologies. Mutants have been created either by treatment of the organism using mutagenic chemical compounds or by repeated passage of the organism in vitro. However, use of either method gives rise to attenuated strains in which the mode of attenuation is unclear. These strains are particularly difficult to characterize in terms of possi...

AI Technical Summary

Benefits of technology

[0007]One such gene identified through this work is surA. The surA gene product is known to promote the folding of extracytoplasmic proteins. Accordingly, the invention provides a vaccine comprising a pharmaceutically acceptable carrier or diluent and a bacterium attenuated by a non-reverting mutation in a gene encoding a protein which promotes the folding of extracytoplasmic proteins. The vaccine has the ability to confer protection against a homologous wild type oral challenge with the virulent bacterium. In addition, the bacterium used in the vaccine can act as a carrier for heterologous antigens such as fragment C of tetanus toxin.DETAILED DESCRIPTION OF THE INVENTIONProteins that Promote the Folding of Extracytoplasmic Proteins

[0016]Infections of ETEC are the single most frequent cause of travellers diarrhoea, causing 3-9 million cases per year amongst visitors to developing countries. In endemic areas, ETEC infections are an important cause of dehydrating diarrhoea in infants and young children, resulting in 800,000 deaths a year in the under fives world-wide. In developing countries, the incidence of ETEC infections leading to clinical disease decreases with age, indicating that immunity to ETEC infection can be acquired. In contrast, naive adults from industrialized countries who visit endemic areas are highly susceptible to ETEC infections. However, with prolonged or repeated visits to endemic areas susceptibility to ETEC infections diminishes, suggesting that a live attenuated approach to ETEC vaccination may prove is successful.

[0018]The bacteria used in vaccines of the invention preferably contain a mutation in one or more genes in addition to the mutation in the gene encoding a protein which promotes folding of extracytoplasmic proteins. This is so that the risk of the bacterium reverting to the virulent state is minimised which is clearly important for the use of the bacterium as a human or animal vaccine. Although bacteria containing only a mutation in a protein which promotes folding of extracytoplasmic proteins are attenuated and the risk of reversion is small, it will generally be desirable to introduce at least one further mutation so as to reduce the risk of attenuation yet further

[0025]The attenuated bacterium used in the vaccine of the invention may be genetically engineered to express an antigen from another organism (a “heterologous antigen”), so that the attenuated bacterium acts as a carrier of the antigen from the other organism. In this way it is possible to create a vaccine which provides protection against the other organism. A multivalent vaccine may be produced which not only provides immunity against the virulent parent of the attenuated bacterium but also provides immunity against the other organism. Furthermore, the attenuated bacterium may be engineered to express more than one heterologous antigen, in which case the heterologous antigens may be from the same or different organisms.

Problems solved by technology

However, use of either method gives rise to attenuated strains in which the mode of attenuation is unclear.

These strains are particularly difficult to characterize in terms of possible reversion to the wild type strain as attenuation may reflect single (easily reversible) or multiple mutation events.

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